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InvisibleInocuole
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Anastomosis
    #23572992 - 08/24/16 02:24 PM (7 years, 5 months ago)

Too many non-advanced topics in Advanced myco. Let's talk about something slightly more advanced than "gargantuous monotubs" or "is this contaminated"?

What I'd really like is to see some pictures, and discussion of, anastomosis. For those who don't know, or don't like using google, anastomosis is the process by which the same, or different hyphae, fuse together. When this happens the individual stands begin to share genetic information with one another. This is an important concept when breeding to select desired traits, because if you are able to identify where anastomosis has occurred, you will more easily be able to select growth which is likely to share traits from its constituent spores/hyphae. ( https://en.wikipedia.org/wiki/Anastomosis )

Problem is, not many people do this kind of work, and even fewer do it publicly. As such, there are basically no pictures of anastomosis occurring with psilocybe mycelium. I don't know how many of you are working on strain-crossing projects currently, but if anyone has any pictures of anastomosis occurring on agar, I would be VERY interested to see them posted here. The more awareness surrounding this process, the less likely that incredible rare genetics will slip by people unnoticed, or be thrown away in unused agar plates.

I want to see this hobby take another huge step forward, and I think getting some more accessible information on this phenomenon in particular will help pave the way. If nobody has any pictures I'll keep trying until I get something and share my own pictures. Either way, let's get some discussion going and figure this shit out.


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Re: Anastomosis [Re: Inocuole]
    #23574584 - 08/24/16 11:43 PM (7 years, 5 months ago)

It's a very good topic Inocuole and I too am getting tired of the off topic threads in this forum.

I wonder if it would be possible to stain one or both of the cultures to make it easy to differentiate between the two and then do time lapse photography through the microscope in order to observe anastomosis take place.


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Re: Anastomosis [Re: inski]
    #23574592 - 08/24/16 11:45 PM (7 years, 5 months ago)

That's a really good idea...  problem I can see maybe encountering is that the process of staining the mycelium with something it can't quickly metabolize may slow it down.  Last time I used really dark food-colored agar, the mycelium took on that hue, and was a bit slower than usual.


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Re: Anastomosis [Re: Inocuole]
    #23574612 - 08/24/16 11:55 PM (7 years, 5 months ago)

Yes, i think the type of stain would be very important and some research and experimentation would be needed to find the right ones.

Regarding food colouring in agar, I purchased some black food colouring and found that it contained two preservatives, (211) and (202).
211 is Sodium benzoate.
202 is Potassium sorbate.

These two are the likely cause of the slow growth in your cultures on agar containing food colouring.


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OfflineGreg
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Re: Anastomosis [Re: inski]
    #23581794 - 08/26/16 09:08 PM (7 years, 5 months ago)

I have nice time-lapse gear and a 40x-2500x microscope.
I also have congo red stain which seems to dye cube myc nicely, not sure how it affects growth though.

I've been considering doing some timelapses of spores germinating, but I've had trouble with tiny vibrations making the captured images blurry.

If someone could capture this process (anastomosis) happening though that would be very interesting.


Edited by Greg (08/26/16 09:22 PM)


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Invisiblec10h12n2o
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Re: Anastomosis [Re: Greg]
    #23601830 - 09/01/16 04:52 PM (7 years, 4 months ago)

Wow, killer topic buddy, thanks for the great info as usual

I'm glad you put a word to this concept for me, I've been trying to learn more about this kind of thing and knowing the right terms will be tremendously useful while I am digging through academic databases :smile:

Is anastomosis what's going on here? I was throwing out some old plates and saw something crazy on one, never seen growth like this. My assumption was a reaction to some kind of bacteria or contam myc, but the thought of strains converging/exchanging crossed my mind



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Re: Anastomosis [Re: c10h12n2o]
    #23601841 - 09/01/16 04:55 PM (7 years, 4 months ago)

That might be contaminated, but yes, anastomosis IS going on there.  I just can't say for sure that it's not the contam that's experiencing the anastomosis.


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Re: Anastomosis [Re: Inocuole]
    #23608304 - 09/03/16 02:53 PM (7 years, 4 months ago)

I'm having trouble understanding where should I look for more info?


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InvisibleInocuole
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Re: Anastomosis [Re: Terpfreak]
    #23608375 - 09/03/16 03:13 PM (7 years, 4 months ago)

Well, that's kind of the point of the thread.  I don't see much info on it, would like people with experience to offer some input.  If before that happens, I happen to be able to discern some things, then I'll certainly share those results.


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Re: Anastomosis [Re: Terpfreak]
    #23608449 - 09/03/16 03:44 PM (7 years, 4 months ago)



Edited by Psilosopherr (09/03/16 03:57 PM)


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OfflineTerpfreak
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Re: Anastomosis [Re: Psilosopherr]
    #23608531 - 09/03/16 04:10 PM (7 years, 4 months ago)

Real question here; why focus on michorizae if most fungi does this?


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Re: Anastomosis [Re: Terpfreak]
    #23608549 - 09/03/16 04:15 PM (7 years, 4 months ago)

Inocuole have you heard of carmine for dye? Its a bug thats powdered and is extremely powerful for coloring . Its only red though .


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Re: Anastomosis [Re: Inocuole]
    #23608569 - 09/03/16 04:20 PM (7 years, 4 months ago)

Quote:

Terpfreak said:
I'm having trouble understanding where should I look for more info?



Quote:

Inocuole said:
Well, that's kind of the point of the thread.  I don't see much info on it, would like people with experience to offer some input.  If before that happens, I happen to be able to discern some things, then I'll certainly share those results.




there are several thousand articles published in academic databases on the subject, though most of it pertains to cancer, and other versions of the phenomenon that dont really apply to our hobby

then there is stuff like this that has useful info, but is very dense and a totally different type of fungus, but it shows the type of diversity that can be seen within cultures even very similar cultures

Highly polymorphic in silico-derived microsatellite loci in the potato-infecting fungal pathogen Rhizoctonia solani anastomosis group 3 from the Colombian Andes.
Authors:
FERRUCHO, R. L.
ZALA, M.
ZHANG, Z.
CUBETA, M. A.
GARCIA-DOMINGUEZ, C.
CERESINI, P. C.
Source:
Molecular Ecology Resources. May2009, Vol. 9 Issue 3, p1013-1016. 4p. 1 Chart.

thanks again for sharing this post inocuole, very interesting stuff. i will also share if i find anything interesting

btw i did transfer some of that crazy crackle growth to see if it persists in subsequent plates

Quote:

Terpfreak said:
Real question here; why focus on michorizae if most fungi does this?




thats a valid question, although i am not sure if most fungi does this.

its so bizarre the transformations myc can go through, rhizo, tomentose, linear, and back again. and then this stuff! lots of dynamics to consider. makes me wonder what we might be missing out on by focusing on aggressive root-like growth


Edited by c10h12n2o (09/03/16 04:24 PM)


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InvisibleInocuole
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Re: Anastomosis [Re: Terpfreak]
    #23608590 - 09/03/16 04:26 PM (7 years, 4 months ago)

Quote:

Terpfreak said:
Inocuole have you heard of carmine for dye? Its a bug thats powdered and is extremely powerful for coloring . Its only red though .




I haven't, but one thing I'd be worried about is that the mycelial network doesn't really seem to work like veins with dye.  I've colored some agar black and paid close attention to the absorption of the color into the mycelium and it seemed to spread rather... indiscriminately, I guess I want to say?  That's not quite the same though, so maybe if the dye was very very concentrated and only placed in one tiny insertion point, it could be useful for highlighting what we're looking for.  I can't help but presume that it would just spread out as pink in all directions though.


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Re: Anastomosis [Re: Inocuole] * 1
    #23608616 - 09/03/16 04:33 PM (7 years, 4 months ago)

That's why dyes like congo red that specifically bind to hyphae are so useful.


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Re: Anastomosis [Re: Greg]
    #23608674 - 09/03/16 04:55 PM (7 years, 4 months ago)

i had no idea!!! thats killer info greg :smile:

is congo red a good dye for working with fungi in general?


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Re: Anastomosis [Re: Greg]
    #23608689 - 09/03/16 05:00 PM (7 years, 4 months ago)

Im not sure what most of this means but here take this- sorry if Im de-railing please let me know.


Quote:

Carmine can be used as a staining agent in histology, as a Best's carmine to stain glycogen, mucicarmine to stain acidic mucopolysaccharides, and carmalum to stain cell nuclei. In these applications, it is applied together with a mordant, usually an Al(III) salt.


-wiki


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Re: Anastomosis [Re: Terpfreak]
    #23609375 - 09/03/16 08:57 PM (7 years, 4 months ago)

Kinda unable to post now but I will be back. . .


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Re: Anastomosis [Re: Pastywhyte]
    #23614485 - 09/05/16 11:26 AM (7 years, 4 months ago)

Cool thread.

I remember reading something about adding rattlesnake venom to agar encouraging anastomosis.

Stamets also talks about serially diluting spore solution so that you can germinate single spores and isolate the cultures before they fuse with other nearby single strains. So if I remember what he wrote correctly, you can put 2 cultures from single, unfused, strains on the same plate. IIRC this is basically the sexual reproduction method of mushrooms, and normally when you streak some spores on a plate this happens when nearby spores germinate and converge. But, if the spores are germinated at a sufficient distance from each other that you can take a wedge before it meets another culture, this is a good way to create hybrids.


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Re: Anastomosis [Re: NumeroEno]
    #23614848 - 09/05/16 01:39 PM (7 years, 4 months ago)

:standingby:


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Re: Anastomosis [Re: Inocuole]
    #23614937 - 09/05/16 02:10 PM (7 years, 4 months ago)

Hi,
Malachite green (Aquarium shop) and Lactophenol blue (microscope outlets) are both pretty cheap and excellent at staining chitin, the major element in fungal cell walls. Problem is they most probably kill the mycelium at a staining strength!, dark field microscopy could be good for live mycelial growth if you can apapt your scope. I've read somewhere about mycelium being grown in a medium on a slide itself/ between slides? can't remember :wink: keep up the good work!


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Re: Anastomosis [Re: wormpoo]
    #23615121 - 09/05/16 03:25 PM (7 years, 4 months ago)

I can't remember if what I wrote about was from TMC or GGMM. I lent my copy of TMC to a friend, and I'm house sitting so away from my copy of GGMM, but next time I go home I'll see if I can post an excerpt.


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Re: Anastomosis [Re: NumeroEno]
    #23615350 - 09/05/16 04:52 PM (7 years, 4 months ago)

I just read GGMM cover to cover and don't remember seeing anything about individual spores. just an fyi


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Re: Anastomosis [Re: Psilosopherr]
    #23616485 - 09/05/16 10:20 PM (7 years, 4 months ago)



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Re: Anastomosis [Re: NumeroEno]
    #23618368 - 09/06/16 01:59 PM (7 years, 4 months ago)

Praise him.


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Re: Anastomosis [Re: Terpfreak]
    #23663307 - 09/20/16 09:34 PM (7 years, 4 months ago)

Im in on this wanna read more but busy


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Re: Anastomosis [Re: Boogieman47]
    #23664078 - 09/21/16 04:02 AM (7 years, 4 months ago)

:thumbup:


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Re: Anastomosis [Re: wtfcrazymofo]
    #23664615 - 09/21/16 10:23 AM (7 years, 4 months ago)

:huxleyfacepalm:


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Re: Anastomosis [Re: Psilosopherr]
    #23665195 - 09/21/16 02:14 PM (7 years, 4 months ago)

ive been trying to learn more about this lately, since i am fascinated by the dynamics of strain organization and interaction in a multi-strain culture. and any genetic exchange at this point in the lifecycle is damn interesting

i was about to throw away a few hundred plates, so i went through them individually to look for anything of interest, and noticed a particular variety has a strong propensity towards developing that crackle looking pattern i posted a pic of earlier in the thread. it has mainly been that same race (AA ), and it takes a while to start developing

I was able to find about a dozen examples, and will photograph them shortly and post pics for talking points. soon i will have a microscope set up to take time lapse videos, and maybe i will be able to catch it during formation

i dont know how much or if it is indeed anastomosis involved, but it seems likely. at first i thought it must be some kind of reaction to bacteria, but the plates seem clean... idk


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Re: Anastomosis [Re: c10h12n2o]
    #23674199 - 09/24/16 02:37 PM (7 years, 4 months ago)

let this one go a bit too long is this anastomosis?


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Re: Anastomosis [Re: Boogieman47]
    #23674571 - 09/24/16 04:53 PM (7 years, 4 months ago)

Nah anastomosis is the process of monokaryotic cultures mating, or in some cases, dikaryotic isolates mating to form a third culture. RR did that with rattlesnake venom to produce PE6. You can't see it happen without a microscope, unless you do it like RR did and a third sector appears at the point where the 2 single sector isolates meet. What you have there is the mycelium that gives way to fruit body formation (the stuff that looks kind of like chain lightning). The anastomosis all happened when the individual monokaryons mated right after the spores germinated.


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Re: Anastomosis [Re: NumeroEno] * 1
    #23674583 - 09/24/16 04:57 PM (7 years, 4 months ago)

Oh ok cool everything about this is interesting as fuck i doubt ill ever get that deep into it but just learning everyday new shit is cool and i really enjoy it


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Re: Anastomosis [Re: Boogieman47]
    #23674617 - 09/24/16 05:05 PM (7 years, 4 months ago)

You seem to be pretty serious about cultivation and a quick learner. Definitely check out the PDF of the Stamets book I posted. That will teach you a lot. You'll be surprised how much information you'll absorb just from being active on the forum.

Anyway, if you want to do your own anastomosis experiment, take a swab of PE, take a swab, of a regular cube, streak them over each other on the same plate, and take a transfer from the last part of your streak. There's a chance you'll get a hybrid that way. It's been done before. You can also do it with a serial dilution of spore solution. If you dilute it enough you can get monokaryotic cultures and mate them on a new plate.


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:gd_icon:  Let it grow! Let it grow! Greatly yield!  :gd_icon:
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Re: Anastomosis [Re: NumeroEno]
    #23674633 - 09/24/16 05:08 PM (7 years, 4 months ago)

Ya im taking my star gazers and pe to some tgis weekend i will read that i appreciate i have to drive a few hours will talk later friend


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Re: Anastomosis [Re: Boogieman47]
    #23674771 - 09/24/16 06:01 PM (7 years, 4 months ago)

To be fair there probably is some anastomosis happening in that plate but as Eno said we can't see it without a scope.

My trials with rustywhyte have all depended on anastomosis to some degree.


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Re: Anastomosis [Re: NumeroEno]
    #23674782 - 09/24/16 06:08 PM (7 years, 4 months ago)

Quote:

NumeroEno said:
You seem to be pretty serious about cultivation and a quick learner. Definitely check out the PDF of the Stamets book I posted. That will teach you a lot. You'll be surprised how much information you'll absorb just from being active on the forum.

Anyway, if you want to do your own anastomosis experiment, take a swab of PE, take a swab, of a regular cube, streak them over each other on the same plate, and take a transfer from the last part of your streak. There's a chance you'll get a hybrid that way. It's been done before. You can also do it with a serial dilution of spore solution. If you dilute it enough you can get monokaryotic cultures and mate them on a new plate.



How would this be a hybrid?  I guess in the sense of strains but not in the sense of species.  That is a little more interesting to me but I don't have any experience myself.  Some interesting literature out there tho.


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Re: Anastomosis [Re: drake89]
    #23674870 - 09/24/16 06:49 PM (7 years, 4 months ago)

It wouldn't be a species hybrid but cube hobbiests refer to varietal crosses as hybrids as do cube spore vendors. It's technically incorrect nomenclature but such things are slow to change in our hobby.


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Re: Anastomosis [Re: Pastywhyte]
    #23675085 - 09/24/16 08:00 PM (7 years, 4 months ago)

:whathesaid:

Crossing 2 varieties of landrace Cannabis indica is also called a hybrid, for example.


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Re: Anastomosis [Re: NumeroEno]
    #23675221 - 09/24/16 08:50 PM (7 years, 4 months ago)

I think as long as you specify whether it's a "strain" or interspecies hybrid, it negates the need to find a new word.


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Re: Anastomosis [Re: Inocuole]
    #23675573 - 09/24/16 11:48 PM (7 years, 4 months ago)

How come taking fresh spores is the best way rather then mixing colonized spawn? Is it just because you can see the growth better that way?


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Re: Anastomosis [Re: Boogieman47]
    #23675578 - 09/24/16 11:53 PM (7 years, 4 months ago)

Because having the psoibily of monokaryns anastomsing or mating together is better for the purpos of creating a varital cross. You can't see it at all unless under a scope.


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Re: Anastomosis [Re: Pastywhyte]
    #23675580 - 09/24/16 11:55 PM (7 years, 4 months ago)

I like how when you get drunk you don't necessarily get any worse at explaining stuff succinctly. :lol:


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Re: Anastomosis [Re: Inocuole]
    #23675584 - 09/24/16 11:58 PM (7 years, 4 months ago)

Lol yer right righttn

K I'm dune. Gnithth


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Re: Anastomosis [Re: Pastywhyte]
    #23675586 - 09/24/16 11:59 PM (7 years, 4 months ago)

Thanks for popping in here dude.  :thumbup:  Will wait til later to ask some specific questions after I brew on em for a bit.


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Re: Anastomosis [Re: Inocuole]
    #23676747 - 09/25/16 01:13 PM (7 years, 4 months ago)

Haha i was trippin last night trying to make out the text and figured i would also wait to respond ... so it would probably be best to use a microscope when trying to cross? Unlesd you just have to guess on cuts


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Re: Anastomosis [Re: Boogieman47]
    #23676824 - 09/25/16 01:43 PM (7 years, 4 months ago)

Quote:

noob47 said:
How come taking fresh spores is the best way rather then mixing colonized spawn? Is it just because you can see the growth better that way?




Colonized spawn consists of already dikaryotic mycelium. If you mix two different types of colonized spawn and fruit it, you'll just get two different types of mushrooms. Try putting transfers from two different cultures on the same plate and watch what happens when they grow against each other. The same thing happens if you mix two different types of spawn.


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Re: Anastomosis [Re: NumeroEno]
    #23676842 - 09/25/16 01:51 PM (7 years, 4 months ago)

So basically once the spores germinate, then trying to get them to fuse is like sticking a square through a circle??... so anastomosis is where they come together?


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Re: Anastomosis [Re: Boogieman47]
    #23676867 - 09/25/16 01:58 PM (7 years, 4 months ago)

If you get enough different strains on converging paths eventually it's likely some will be able to fuse and many should be able to fruit together. Serial dilutions and monokaryons are handy but not completely required for a cube ir varietal cross. If you can use fresh spores it will be likely some monokaryons will mate as well.


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Re: Anastomosis [Re: Pastywhyte]
    #23676993 - 09/25/16 02:49 PM (7 years, 4 months ago)

Ok that makes sense im swabbing my plate tonight


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Re: Anastomosis [Re: Inocuole]
    #23677692 - 09/25/16 07:13 PM (7 years, 4 months ago)

+5


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Re: Anastomosis [Re: Duke of disjour]
    #23682309 - 09/27/16 06:51 AM (7 years, 4 months ago)

:takingnotes:


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Re: Anastomosis [Re: catnip40]
    #23701627 - 10/02/16 08:57 PM (7 years, 3 months ago)

So, some of us were talking on another forum about how mycelium can intertwine and share characteristics; eg. Mycogone growing from cube substrate can bruise, and the oysters that invaded Pussyfart's cubensis cakes would bruise (but weren't active).

Is this all anastomosis of different species? Just how much of these characteristics can actually be shared, I wonder. Considering that an invading mold can take on the bruising characteristic when growing on Cubensis mycelium, I find that really amazing. Mad Season speculated it was from the digestion of the cubensis mycelium by the invading mycelium, which seems completely plausible. But say in the case of the oysters, their bluing didn't make them active at all (if bluing were to actually be deterioration of Psilocin, which we know is probably not true).

I don't really have a proper question formed just yet. Just thinking out loud here.
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edit: goddamn it. Both PF and Muda got oyster infested cubes, but Muda's bruised. Sorry for the mixup.


Edited by dankington (10/03/16 07:26 AM)


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Re: Anastomosis [Re: dankington]
    #23701639 - 10/02/16 09:00 PM (7 years, 3 months ago)

One thing about white molds is that the nasty disgusting blobs they produce are indeed cube fruits. That's why they bruise blue. You can eat them and trip but it's nasty shit.


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Re: Anastomosis [Re: Pastywhyte]
    #23702354 - 10/03/16 03:11 AM (7 years, 3 months ago)

I don't think the absorption of the bluing mechanism is a sign of anastomosis.  If anything it's just a sign that the competing colony was able to absorb some of the enzymes and chemicals produced by the mycelium.

Also, it was Muda whose oysters turned blue.  So now you've gone and credited PF for that in multiple threads. :lol:


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Re: Anastomosis [Re: Inocuole]
    #23702602 - 10/03/16 07:01 AM (7 years, 3 months ago)

:smbfacepalm:
I guess I got a little mixed up. PF did just get oysters in with his cubes, didn't mention the bluing
https://www.shroomery.org/forums/showflat.php/Number/23205057#23205057

Here's muda's blue oysters


Edited by dankington (10/03/16 08:38 AM)


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Re: Anastomosis [Re: dankington]
    #23705896 - 10/04/16 07:17 AM (7 years, 3 months ago)

Well I thought I would get a few pics before I start cloning and cutting this plate up. The big section with all the little pins is a crs culture that's pretty old and all it does anymore is pin. The small section is a lucy crs/aa+ f4 cross not rust spored that came apart . I'm planning on cloning the big pins on the party line and see what they are .I like waiting on the plates to pin then look for hybrid vigor on the party lines.


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Re: Anastomosis [Re: Inocuole]
    #23846515 - 11/18/16 08:14 PM (7 years, 2 months ago)

Couple colonies from syringe.  Anastomosis over the zone of inhibition?


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